Novel stable PACAP analogs with potent activity towards the PAC1 receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid neuropeptide with promising therapeutic applications for the treatment of several pathophysiological states related to neurodegenerative diseases. However, its use for therapeutic applications is actually limited by its restricted bioavailability and rapid degradation. Therefore, metabolically stable PACAP analogs represent promising tools to further investigate the physiological roles of PACAP and ascertain its usefulness in some clinical conditions. In this study, derivatives of PACAP27 and PACAP38 have been rationally designed to develop PAC1 receptor agonists resistant to peptidase action. Results showed that N-terminal modifications confer resistance to dipeptidyl peptidase IV, a major proteolytic process involved in PACAP degradation. Moreover, in vitro incubation of both PACAP isoforms in human plasma revealed that PACAP38 is rapidly metabolized, with a half-life of less than 5 min, while PACAP27 was stable in these experimental conditions. Hence, following the elucidation of its plasmatic metabolites, PACAP38 was modified at its putative endopeptidase and carboxypeptidase sites of cleavage. All peptide analogs were tested for their ability to bind the PAC1 receptor, as well as for their potency to induce calcium mobilization and inhibit PC12 cell proliferation through the PAC1 receptor. This approach revealed two leading compounds, i.e. acetyl-[Ala15, Ala20]PACAP38-propylamide and acetyl-PACAP27-propylamide, which exhibited improved metabolic stability and potent biological activity. This study describes innovative data related to PACAP metabolism in human plasma and depicts the development of a metabolically stable PACAP38 analog, acetyl-[Ala15, Ala20]PACAP38-propylamide, which behaves as a super-agonist towards the PAC1 receptor.

Photic induction of c-Fos in enkephalin neurons of the rat intergeniculate leaflet innervated by retinal PACAP fibres.

The brain's biological clock, located in the suprachiasmatic nucleus (SCN), is synchronised with the cyclic environment by photic and non-photic cues. Photic information to the SCN is mediated by pituitary adenylate-cyclase-activating polypeptide (PACAP)-containing retinal ganglion cells (RGCs), whereas non-photic input originates primarily from neuropeptide Y (NPY) cells in the ipsilateral thalamic intergeniculate leaflet (IGL). RGCs also seem to project to the IGL, indicating a role for this structure in the integration of photic and non-photic inputs related to the resetting of the biological clock. In the present study, we have used anterograde tracing from both eyes, bilateral eye enucleation, double-immunofluorescence histochemistry, high-resolution confocal laser scanning microscopy and three-dimensional computer analysis to show that (1) PACAP-containing RGCs project to the IGL and are the only source for the PACAP-immunoreactive fibres in the IGL; (2) a few NPY-containing neurons in the IGL are innervated by PACAP-containing retinal nerve fibres and the contacts are both axodendritic and axosomatic; (3) most enkephalin-immunoreactive neurons in the IGL are innervated by PACAP-containing retinal afferents and the contacts are mainly axodendritic; (4) light stimulation at various time points activates (as evidenced by c-Fos induction) enkephalin-positive neurons but not NPY-immunoreactive neurons. The findings suggest that PACAP-immunoreactive retinal afferents in the IGL primarily innervate enkephalin-immunoactive neurons and that the enkephalin-containing neurons, which project locally and to the contralateral IGL, are activated by light independent of diurnal time.